Project summary/Abstract: Treatment with chemotherapeutic agents can have lasting effects not only on female fertility, but also on normal hormonal homeostasis, by triggering premature menopause. Chemotherapeutics can be particularly devastating to girls who are too young to have embryo or egg freezing as a fertility-preserving option. Currently no treatment exists which can both protect the ovaries in situ, and maintain long-term hormonal function. We have recently demonstrated that Mullerian Inhibiting Substance (MIS) is a powerful ovarian suppressant which can protect both the ovary and the uterus against the damages of chemotherapy. However, the mechanisms of protection of these organs by MIS remain unknown. Our long-term goal is to understand how chemotherapy damages fertility, and which protective pathways are elicited by MIS in the ovary and uterus. These discoveries could facilitate the development of novel drugs that can protect both fertility and endocrine function in female patients undergoing chemotherapy. We hypothesize that MIS, or agonists of its receptor, can protect the ovarian reserve, prevent uterine dystocia, and reduce the risk of premature ovarian insufficiency associated with chemotherapy. Our rationale is that MIS is the only known hormone capable of blocking primordial follicle activation, and thus understanding this pathway could identify an entirely new class of therapeutic targets. Our specific aims will test the following hypotheses: 1) that MIS protects the ovary from chemotherapy by inhibiting primordial follicle activation and granulosa cell apoptosis; 2) that MIS prevents uterine labor dystocia caused by doxorubicin by modulating uterine tissue repair; and 3) that newly identified agonists of the MIS receptor can be shown to preserve fertility in mice treated with chemotherapy. The proposed studies represent a significant contribution to our understanding of the mechanisms of action of MIS in fertoprotection, with a potential for significant clinical impact. Furthermore, they will provide novel insights into the mechanism of follicle dormancy, one of the greatest remaining mysteries of reproductive biology. These studies are highly innovative by providing for the first time a comprehensive assessment of how chemotherapy damages the ovary and uterus at the single cell resolution through the use of single cell transcriptomics. Thus, MIS and its small molecule analogs may provide a novel paradigm of a contraceptive that can protect both ovarian and uterine function in female patients undergoing chemotherapy.